Amorphous Calcium Carbonate Precipitation by Cellular Biomineralization in Mantle Cell Cultures of Pinctada fucata

Abstract

The growth of molluscan shell crystals is generally thought to be initiated from the extrapallial fluid by matrix proteins, however, the cellular mechanisms of shell formation pathway remain unknown. Here, we first report amorphous calcium carbonate (ACC) precipitation by cellular biomineralization in primary mantle cell cultures of Pinctada fucata. Through real-time PCR and western blot analyses, we demonstrate that mantle cells retain the ability to synthesize and secrete ACCBP, Pif80 and nacrein in vitro. In addition, the cells also maintained high levels of alkaline phosphatase and carbonic anhydrase activity, enzymes responsible for shell formation. On the basis of polarized light microscopy and scanning electron microscopy, we observed intracellular crystals production by mantle cells in vitro. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed the crystals to be ACC, and de novo biomineralization was confirmed by following the incorporation of Sr into calcium carbonate. Our results demonstrate the ability of mantle cells to perform fundamental biomineralization processes via amorphous calcium carbonate, and these cells may be directly involved in pearl oyster shell formation.

Calcium carbonate mineralization mediated by in vitro cultured mantle cells from Pinctada fucata

“…Some particles adhered on the smooth surfaces of the crystals. The round particles with the black arrow had a 10-mm diameter, which was similar to the size of mantle cells reported previously [17]. The results of the EDS analysis of the crystal and the two round particles suspected to be mantle cells are shown in Fig.…”

“…However, whether mantle cells are directly involved in the mineralization process remains unknown. It has been demonstrated previously that in vitro cultured mantle cells retain their ability to secrete mineralization-related enzymes and matrix proteins that can be detected in the cultured medium [17]. However, in our study, no calcium carbonate crystals grew in cultured medium containing 25 mM Ca 2þ (Fig.…”

“…In our study, calcium carbonate crystals only appeared when mantle cells were present, strongly suggesting that mantle cells are responsible for the crystal nucleation process. This suggestion can be confirmed by the existence of ACC crystals in mantle cells [17]. ACC is a highly disordered phase and a precursor of calcite and aragonite [20,21].…”

“…A saturated calcium bicarbonate solution was prepared according to the method of Xiang et al [17]. Twelve-day-old medium that had been used in cultures was collected and filtered through a 0.22-mm membrane filter to remove cells and insoluble substances.…”

“…Furthermore, it was demonstrated that mantle cells still have the ability to secrete matrix proteins in primary cell culture. Xiang et al was also the first to report the existence of amorphous calcium carbonate (ACC) inside mantle cells [17]. Considering the remarkable contribution of mantle cells to shell formation, it is essential to establish in vitro mineralization models to research the related cellular mechanisms.…”